Project Summary - Project 2 (PD: Pamela Bjorkman) Zika virus (ZIKV) infection is an emerging global health concern due to the potential for fetal abnormalities when infection occurs during pregnancy. A ZIKV vaccine should preferentially elicit neutralizing antibodies (Abs) rather than non-neutralizing Abs, which may exacerbate disease caused by ZIKV or related dengue virus strains through the phenomenon of Ab-dependent enhancement (ADE). The Bjorkman lab proposes to use knowledge of the structural basis for Ab neutralization of ZIKV to design potential vaccine immunogens optimized to elicit neutralizing Abs while reducing the risk of ADE by minimizing the production of non- neutralizing Abs. The Nussenzweig, Rice, and Bjorkman laboratories have recently-established a collaboration to isolate and characterize Abs against ZIKV Envelope Domain III (ZEDIII). A group of ZIKV-infected individuals with high ZIKV neutralizing Ab activity have been found to have Abs derived from immunoglobulin genes VH3- 23/VK1-5. Several of these Abs neutralize ZIKV in vivo and are effective in challenge and in treatment experiments in mice. The structural basis for ZIKV neutralization by these Abs has been revealed by the Bjorkman lab in crystal structures of Ab?EDIII complexes. The crystal structures of these Abs bound to ZEDIII and to the counterpart domain of dengue 1 virus (DENV1) revealed a common mechanism of recognition of the ZIKV and DENV1 EDIII lateral ridge. The Bjorkman lab will extend these efforts to solve and compare structures of additional neutralizing Abs bound to their target antigens, both isolated envelope domains and virions, in order to determine which features correlate with neutralizing activity. Complexes of antigen with neutralizing Abs of varying cross-reactivity and potency (isolated by Dr. Nussenzweig and evaluated by Dr. Rice) will be crystallized and their structural features compared to identify viral vulnerabilities as well as characterize epitopes targeted by weak or non-neutralizing antibodies. The Bjorkman lab proposes to use the germline-targeting approach for ZIKV immunogen design to mitigate the potential for ADE, an approach made possible by the identification and characterization of the VH3-23/VK1-5 class of anti-ZIKV Abs. Initial immunogen design efforts will be focused on the lateral ridge epitope, which is recognized by the potent VH3-23/VK1-5 Abs. Yeast library screening methods successfully used for HIV-1 immunogen design will be adapted to identify ZEDIII variants that bind with higher affinity to iGL versions of VH3-23/VK1-5 Abs. Non-neutralizing epitopes will be masked by adding N-linked glycans or altered to reduce immunogenicity. Optimized antigens will be multimerized to generate candidate immunogens. These immunogens will be evaluated for efficacy and safety in pre-clinical models in collaboration with Drs. Nussenzweig and Rice. The goal is to develop immunogens suitable to move towards clinical testing.